1. Field of the Invention
The invention relates to a plasmid replication origin, and to plasmids containing the said origin.
2. Discussion of the Background
Plasmids are extrachromosomal circular DNA fragments which are transferable from one bacterium to another, and whose replication occurs independently of that of the bacterial chromosome. A given plasmid can be present in a high copy number inside a bacterial cell. The copy number is a genetic characteristic of each plasmid. For example, in the ColE1-type plasmids (such as plasmids of the families pBR, pUC, and the like), the copy number is under the control of a DNA region corresponding to the replication origin of the plasmid (ORI) which extends approximately between bases 2940 and 3130 (numbering of the bases of pBR322 proposed by PEDEN [Gene, 22 (1983) 277-280]). A portion of this region, situated between bases 2970 and 3089 is transcribed into RNAs called RNAI and RNAII. RNAI, in particular, is thought to play a role in the regulation of the plasmid copy number.
Plasmids are commonly used in genetic engineering as vectors for the cloning and expression of foreign genes in bacteria.
It is particularly desirable, for this purpose, to have available plasmids present in high copy number, either in order to obtain the foreign DNA in a large quantity (for example in order to allow its sequencing), or in order to increase the quantity of the product of expression of the said foreign gene.
A conventional technique for obtaining a large quantity of plasmids is to add to the bacteria cultures chloramphenicol which prevents cell multiplication by blocking translation, without affecting plasmid replication. Nevertheless, since chloramphenicol also interferes with the translation of the foreign gene, this method is not optimal when an increase in the expression of the said gene is desired.
Plasmids carrying a mutation which influences the copy number have been described in the literature. BOROS et al. [Gene, 30, 257-260 (1984)] thus describe a mutant plasmid derived from pBR322. The copy number of this plasmid per cell is increased by about 200-fold relative to the copy number of pBR322. This increase in the number of copies results from a G to T transversion localized in position 3075 on the 2846-3363 HinfI fragment, close to the 3' end of the sequence transcribed into RNAI. MUESINO et al. [Cell, 24, 235-242, (1981)] had earlier demonstrated the same mutation in the plasmid ColE1 (whose replication origin is similar through the sequence to that of pBR322), also with, as effect, an increase in the copy number of the said plasmid (up to 300 per cell).
However, such an amplification of the copy number has certain disadvantages which limit its practical applications. This indeed results in:
an increased risk of toxicity for the host bacterium, by too high an expression of the recombinant genes carried by the plasmid, which is in addition generally accompanied by the production of precipitation granules and makes it impossible, in addition, to envisage the expression of periplasmic proteins;
an increased probability of uncontrolled recombinations between plasmids, in particular for the plasmids carrying certain specific sequences (for example of phage origin).
Now, during experiments for mutagenesis of the plasmid pUC9, the inventors have obtained plasmids carrying mutations in the ORI sequence of the replication origin, and have observed that, surprisingly, the copy number of these plasmids is 10 to 25-fold higher than that of the original plasmid.
The inventors have sequenced the replication origins of the mutated plasmids, and have localized the corresponding mutations.